Energetics and dynamics of the FOXP2 forkhead domain-DNA interaction

Abstract

The members of the forkhead box (FOX) family of transcription factors are key regulators in the development and metabolism of a wide variety of tissues in humans. The FOX transcription factors are classified by the presence of a canonical forkhead winged-helix DNA binding domain and are further divided into subfamilies based on sequence divergence of the forkhead domain. To date, only the FOXP subfamily is known to require dimerisation for transcriptional activity. Dimerisation of the FOXP subfamily members occurs at two distinct interfaces, a conserved leucine zipper domain, and through domain-swapping of a C-terminal forkhead domain. The role of the domain-swapped forkhead domain is unclear, although several attempts have been made to clarify this. Due to the orientation of the recognition motifs in the domain-swapped dimer, it has been speculated that it is capable of binding and congregating two distal promoter response elements, suggesting a role in cross-chromosomal gene co-regulation. The unique capability of the FOXP forkhead domain to dimerise is attributed to an evolutionary mutation (proline to alanine) that occurs in the hinge loop connecting the second and third α-helices. Further to this, the hinge loop has also been implicated in altering the specificity of the forkhead domain. Here, we aim to elucidate how the evolutionary proline to alanine mutation facilitates dimerisation and whether it has any role in defining the DNA binding specificity of the FOXP2 forkhead domain. To do this all experiments were conducted on an obligate monomeric mutant (A539P) and an engineered obligate dimeric mutant (F541C) FOXP2 forkhead domain in addition to the wild-type. High and low-resolution DNA binding studies involving electrophoretic mobility shift assays (EMSA), fluorescence polarisation (FP) studies and isothermal titration calorimetry (ITC) revealed that the FOXP2 forkhead domain preferentially binds to the FOXP2 consensus site as a monomer, despite having the capacity to form dimers in the absence of DNA. During these studies, a significant difference in the thermodynamic signatures of DNA binding was observed between the wild-type and A539P mutant FOXP2 forkhead domain. Further dissection of the

thermodynamic results revealed that the hinge loop mutation significantly alters the mechanism of DNA binding. The wild-type FOXP2 forkhead domain undergoes significant conformational changes upon DNA binding, shown by hydrogen-deuterium exchange mass spectrometry, in addition to making two additional contacts with the sugar-phosphate backbone of the consensus site. The large conformational changes incurred by DNA binding, stabilises the monomeric form of the FOXP2 forkhead domain and is indicative of a searchrecognition conformational switch that is unique to the FOXP subfamily. Furthermore, in vivo studies, involving dual-luciferase reporter assays, show that dimerisation of the FOXP2 forkhead domain acts as a regulatory mechanism controlling the transcriptional activity of FOXP2. Together the work presented here proposes that the DNA binding by FOXP2, and by extension FOXP1 and 4, follows a monomeric pathway whereby FOXP2 translocate to the site of action as a monomer and in a context-dependent manner either dimerises or remains monomeric to fine-tune the regulation of target genes. This work provides the first detailed assessment of the energetics and dynamics that occur during DNA binding for not only the FOXP2 forkhead domain but any of the FOX forkhead domains. Furthermore, presented here is the first proposed mechanism of transcriptional regulation through the oligomeric state of the FOXP2 forkhead domain.